The quantitative measurement of cytosolic calcium concentration is central to the study of excitation-contraction coupling in cardiac cells. Significant problems, however, complicate the valid calibration of cardiac cells loaded with the membrane-permanent acetoxymethyl ester (AM) forms of the Ca 2+ probes, Indo-1 and Fura-2, notable mitochondrial dye compartmentation and incomplete AM-ester hydrolysis. We have developed a novel, simple and practical method to selectively load Indo-1 "free acid" into the cytosol of adult cardiac myocytes presumable by diffusion through momentarily-permeable gap junctions during mechanical dissociation after low Ca 2+, collagenase treatment. A range of Indo-1 loading has been defined to select cells that produce suitable fluorescence signals, without significant alteration of their functional properties compared to non-Indo loaded cells; in particular, maximal twitch shortening velocity and extend of shortening in this range of Indo-1 is unchanged, with only minimal (less than or equal to 10%) prolongation of twitch duration (i.e., due to Ca 2+ buffering). Calibration of Indo-1 fluorescence in these cells has been accomplished after subtracting average autofluorescence derived from time- matched non-Indo loaded cells. Since there is wide variation in the degree of uncertainty of individual [Ca 2+] i determinations among cells (related principally to differences in cellular Indo-1 content, to nonlinear aspects of the [Ca 2+] vs. fluorescence ratio relationship, and to the uncertainty in the autoflourescence subtraction), a quantitative estimate of uncertainty may also be employed in formulating weighted, valid estimates of cytosolic [Ca 2+]i. The following [Ca 2+] i values in rat ventricular cells (nM; in 1 mM bathing [Ca 2+] o, 25 degrees C) are given as weighted means plus/minus 95% confidence intervals (unweighted values in parenthesis): 138 plus/minus 5 (136 plus/minus 6, n=44) in quiescent cells, 435 plus/minus 74 (482 plus/minus 76, n=43) at the [Ca 2+] i- transient peak during 0.5 Hz steady state stimulation, and 760 plus/minus 124 (1,027 plus/minus 250, n=42) at the [Ca 2+] i-transient peak, post- rest. Thus, the technique affords a practical way to accurately assess [Ca 2+] i in large numbers of isolated cardiac cells from a single batch-loaded preparation, avoiding many of the inherent limitations of micropipette impalement methods (i.e., cytosol dialysis, brief cell survival).